Abstract: Cells retain their identity by inheriting and maintaining gene expression profiles of their predecessors. Patterns of gene expression that propagate through cell division are thought to be established and maintained in part through reversible covalent modifications of DNA and DNA-associated proteins (i.e., histones). These heritable patterns of gene expression do not involve changes in the DNA sequence and are termed 'epigenetic'. Emerging evidence implicates epigenetic inheritance in a myriad of developmental processes as well as being a significant contributor to human diseases. While many epigenetic modifications correlate with specific gene expression states, evidence supporting specific factors in initiating epigenetic control of gene expression remains scant. Finding the molecular basis of epigenetic triggers will elucidate how mitotically- heritable patterns of gene expression are established during development and disease. Moreover, due to the potential reversibility of epigenetic modifications, the identification of epigenetic initiators may provide key candidates for manipulating the transcriptional profile of cells for therapeutic purposes. We propose to comprehensively identify factors and mechanisms that initiate as well as prevent epigenetic transcriptional inactivation, through a groundbreaking approach in the study of X-chromosome inactivation. X-inactivation provides an experimentally tractable system for the dissection of epigenetic inheritance. X-inactivation results in the mitotically-heritable transcriptional inactivation of one X-chromosome in female mammals, thereby equalizing X-linked gene dosage between males and females. The epigenetic factors and mechanisms that execute X-inactivation are known to overlap with those that regulate embryonic development and disease progression (i.e., cancers). Thus, understanding the cascade of epigenetic events that characterizes the inactive- as well as the active-X offers a window into identifying the common factors that initiate and prevent epigenetic transcriptional inactivation during development and disease. Contrary to the prevailing models of X- inactivation initiation, we have established compelling evidence that the factors thought to trigger X- inactivation, Xist RNA and Polycomb group proteins, are both dispensable for the initiation of chromosome- wide X-linked gene silencing during mouse imprinted X-inactivation. We have also gathered data that indicates that the Xist anti-sense transcript Tsix, proposed to forestall inactivation of the active-X, is dispensable in preventing inactivation of the active X-chromosome during the initiation phase of imprinted X- inactivation. Moreover, proteins that prevent the epigenetic transcriptional inactivation of the active-X remain unknown. In sum, these findings imply that undiscovered epigenetic factors must exist to initiate as well as prevent X-inactivation, and is the focus of this proposal. Public Health Relevance: This proposal aims to discover and delineate novel factors and mechanisms that trigger epigenetic transcriptional regulation. Dysregulation of the epigenetic cellular machinery is increasingly being recognized as a cause of, or significant contributor to, human diseases such as cancers. Importantly, as opposed to irreversible mutations in DNA, epigenetic modifications are reversible. This reversibility makes epigenetic changes, including those we propose to identify, potentially amenable to manipulation and therapeutic intervention.